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Cell Reports Nov 2023Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by...
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
Topics: Animals; Dyneins; Biological Transport; Cilia; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Flagella
PubMed: 37883232
DOI: 10.1016/j.celrep.2023.113337 -
The Journal of Biological Chemistry Sep 2023Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory...
Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule in vitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.
Topics: Dyneins; Kinesins; Microtubule-Associated Proteins; Septins; COS Cells; HEK293 Cells; Humans; Animals; Chlorocebus aethiops; Protein Transport
PubMed: 37495111
DOI: 10.1016/j.jbc.2023.105084 -
Cells Feb 2024Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved... (Review)
Review
Dynein, an ancient microtubule-based motor protein, performs diverse cellular functions in nearly all eukaryotic cells, with the exception of land plants. It has evolved into three subfamilies-cytoplasmic dynein-1, cytoplasmic dynein-2, and axonemal dyneins-each differentiated by their cellular functions. These megadalton complexes consist of multiple subunits, with the heavy chain being the largest subunit that generates motion and force along microtubules by converting the chemical energy of ATP hydrolysis into mechanical work. Beyond this catalytic core, the functionality of dynein is significantly enhanced by numerous non-catalytic subunits. These subunits are integral to the complex, contributing to its stability, regulating its enzymatic activities, targeting it to specific cellular locations, and mediating its interactions with other cofactors. The diversity of non-catalytic subunits expands dynein's cellular roles, enabling it to perform critical tasks despite the conservation of its heavy chains. In this review, we discuss recent findings and insights regarding these non-catalytic subunits.
Topics: Cytoplasmic Dyneins; Catalytic Domain; Dyneins
PubMed: 38391943
DOI: 10.3390/cells13040330 -
Frontiers in Endocrinology 2023Primary cilia are microtubule-based organelles that are widespread on the cell surface and play a key role in tissue development and homeostasis by sensing and... (Review)
Review
Primary cilia are microtubule-based organelles that are widespread on the cell surface and play a key role in tissue development and homeostasis by sensing and transducing various signaling pathways. The process of intraflagellar transport (IFT), which is propelled by kinesin and dynein motors, plays a crucial role in the formation and functionality of cilia. Abnormalities in the cilia or ciliary transport system often cause a range of clinical conditions collectively known as ciliopathies, which include polydactyly, short ribs, scoliosis, thoracic stenosis and many abnormalities in the bones and cartilage. In this review, we summarize recent findings on the role of primary cilia and ciliary transport systems in bone development, we describe the role of cilia in bone formation, cartilage development and bone resorption, and we summarize advances in the study of primary cilia in fracture healing. In addition, the recent discovery of crosstalk between integrins and primary cilia provides new insights into how primary cilia affect bone.
Topics: Cilia; Flagella; Biological Transport; Kinesins; Dyneins
PubMed: 37886641
DOI: 10.3389/fendo.2023.1259650 -
Cells Oct 2023Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At...
Axonemal dyneins are highly complex microtubule motors that power ciliary motility. These multi-subunit enzymes are assembled at dedicated sites within the cytoplasm. At least nineteen cytosolic factors are specifically needed to generate dynein holoenzymes and/or for their trafficking to the growing cilium. Many proteins are subject to N-terminal processing and acetylation, which can generate degrons subject to the N-end rule, alter N-terminal electrostatics, generate new binding interfaces, and affect subunit stoichiometry through targeted degradation. Here, we have used mass spectrometry of cilia samples and electrophoretically purified dynein heavy chains from to define their N-terminal processing; we also detail the N-terminal acetylase complexes present in this organism. We identify four classes of dynein heavy chain based on their processing pathways by two distinct acetylases, one of which is dependent on methionine aminopeptidase activity. In addition, we find that one component of both the outer dynein arm intermediate/light chain subcomplex and the docking complex is processed to yield an unmodified Pro residue, which may provide a setpoint to direct the cytosolic stoichiometry of other dynein complex subunits that contain N-terminal degrons. Thus, we identify and describe an additional level of processing and complexity in the pathways leading to axonemal dynein formation in cytoplasm.
Topics: Axonemal Dyneins; Microtubules; Chlamydomonas; Cilia; Axoneme
PubMed: 37887336
DOI: 10.3390/cells12202492 -
The EMBO Journal Apr 2024Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying...
Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.
Topics: Dyneins; Cryoelectron Microscopy; Biological Transport; Cilia; Flagella
PubMed: 38454149
DOI: 10.1038/s44318-024-00060-1 -
Nature Communications Nov 2023Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with...
Intracellular vesicular transport along cytoskeletal filaments ensures targeted cargo delivery. Such transport is rarely unidirectional but rather bidirectional, with frequent directional reversals owing to the simultaneous presence of opposite-polarity motors. So far, it has been unclear whether such complex motility pattern results from the sole mechanical interplay between opposite-polarity motors or requires regulators. Here, we demonstrate that a minimal system, comprising purified Dynein-Dynactin-BICD2 (DDB) and kinesin-3 (KIF16B) attached to large unilamellar vesicles, faithfully reproduces in vivo cargo motility, including runs, pauses, and reversals. Remarkably, opposing motors do not affect vesicle velocity during runs. Our computational model reveals that the engagement of a small number of motors is pivotal for transitioning between runs and pauses. Taken together, our results suggest that motors bound to vesicular cargo transiently engage in a tug-of-war during pauses. Subsequently, stochastic motor attachment and detachment events can lead to directional reversals without the need for regulators.
Topics: Dyneins; Kinesins; Biological Transport; Cytoskeleton; Dynactin Complex; Microtubules
PubMed: 37985763
DOI: 10.1038/s41467-023-42605-8 -
Nature Communications Sep 2023Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the...
Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, using cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localize 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also find that the CCDC96/113 complex is in close contact with the DRC9/10 in the linker region. In addition, we reveal that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.
Topics: Dyneins; Cryoelectron Microscopy; Microtubule-Associated Proteins; Cytoskeleton; Axoneme; Amyloidogenic Proteins
PubMed: 37714832
DOI: 10.1038/s41467-023-41480-7 -
Zn2+ decoration of microtubules arrests axonal transport and displaces tau, doublecortin, and MAP2C.The Journal of Cell Biology Aug 2023Intracellular Zn2+ concentrations increase via depolarization-mediated influx or intracellular release, but the immediate effects of Zn2+ signals on neuron function are...
Intracellular Zn2+ concentrations increase via depolarization-mediated influx or intracellular release, but the immediate effects of Zn2+ signals on neuron function are not fully understood. By simultaneous recording of cytosolic Zn2+ and organelle motility, we find that elevated Zn2+ (IC50 ≈ 5-10 nM) reduces both lysosomal and mitochondrial motility in primary rat hippocampal neurons and HeLa cells. Using live-cell confocal microscopy and in vitro single-molecule TIRF imaging, we reveal that Zn2+ inhibits activity of motor proteins (kinesin and dynein) without disrupting their microtubule binding. Instead, Zn2+ directly binds to microtubules and selectively promotes detachment of tau, DCX, and MAP2C, but not MAP1B, MAP4, MAP7, MAP9, or p150glued. Bioinformatic predictions and structural modeling show that the Zn2+ binding sites on microtubules partially overlap with the microtubule binding sites of tau, DCX, dynein, and kinesin. Our results reveal that intraneuronal Zn2+ regulates axonal transport and microtubule-based processes by interacting with microtubules.
Topics: Animals; Humans; Rats; Axonal Transport; Doublecortin Domain Proteins; Dyneins; HeLa Cells; Kinesins; Microtubule-Associated Proteins; Microtubules; tau Proteins; Zinc
PubMed: 37326602
DOI: 10.1083/jcb.202208121 -
EMBO Reports Apr 2024Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe...
Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.
Topics: Animals; Humans; Male; Mice; Adaptor Proteins, Signal Transducing; Cytoskeletal Proteins; Dyneins; HSC70 Heat-Shock Proteins; Infertility, Male; Molecular Chaperones; Protein Folding; Semen; Sperm Head; Spermatogenesis; Spermatozoa; Teratozoospermia; Thiazoles
PubMed: 38454159
DOI: 10.1038/s44319-024-00112-x